CN102766213B - Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting furaltadone residue marker AMOZ - Google Patents
Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting furaltadone residue marker AMOZ Download PDFInfo
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Abstract
The invention discloses a monoclonal antibody capable of identifying a furaltadone residue marker 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ). The monoclonal antibody is secreted by a hybridoma cell AMOZ; and the hybridoma is preserved in China Center for Type Culture Collection, and has a preservation number of CCTCC NO:C201188. The invention also discloses an enzyme immunoassay method and a reagent kit for detecting furaltadone marker residue marker AMOZ, and application thereof to detection of the furanketone residue marker AMOZ. Compared with prior art, the monoclonal antibody prepared by the invention, the ELISA Kit and the ELISA method have high detection sensitivity, high precision and good accuracy; a hapten synthesis process is simple, and has high synthesis efficiency; and a derivatization reagent for sample pretreatment is benzene formaldehyde, which has advantage of small toxicity compared with other commonly used derivative reagent like o-nitrobenzaldehyde.
Description
Technical field
The invention belongs to detect and analyze and immunological technique field, be specifically related to one and can detect monoclonal antibody and enzyme-linked immunoassay method and the test kit of residue of furaltadone through marker 3-amino-5-beautiful jade for methyl-2-oxazolidone (AMOZ).
Background technology
Furaltadone belongs to itrofurans medicine, has the basic structure of 5-nitrofuran ring, is a kind of broad spectrum antibiotic, all effective to gram-positive microorganism, Gram-negative bacteria, fungi, protozoon etc., once in aquaculture, is widely used.Toxicology test proves that Furaltadone and metabolite thereof all have the serious toxicity effects such as carcinogenic, mutagenesis significantly.European Union forbids that in 1993 Furaltadone uses in aquaculture, and No. 193 bulletins " veterinary drug and other compound inventories of food animal forbidding " that China issues are listed itrofurans medicine in forbidding inventory.
Fast due to Furaltadone low price, drug effect, still have at present the violated use of a lot of raisers.China is still severe to the residual monitoring situation of itrofurans medicine, must improve as early as possible screening detection system, to ensure the public's life and health safety and to promote carrying out smoothly of domestic and international economic trade.
HPLC-MS/MS is the confirmation means of current generally acknowledged itrofurans medicine residue detection, can reach 0.1 ~ 0.3 μ g/kg to the detectability of the samples such as milk, eggs, pork, poultry and shrimp.Although the sensitivity of the method and tolerance range are all very high, need to be equipped with expensive instrument and professional and technical personnel, are not suitable for field operation and high flux screening.
Immunological detection method for high flux screening is the biologically based on antigen-antibody, highly sensitive and easy handling.Umaporn Pimpitak etc. (2009) etc. have prepared the monoclonal antibody of AMOZ, the IC of antibody
50value (taking CPAMOZ calculating concentration) is 0.023 μ g/L, adopts direct competitive ELISA method to detect AMOZ is residual in shrimp sample.But the method detects after needing first AMOZ to be reacted with terephthalaldehydic acid again, the efficiency of reaction is low, can not be widely used.
Summary of the invention
The object of this invention is to provide one and can identify enzyme-linked immunoassay method and the test kit of residue of furaltadone through marker 3-amino-5-beautiful jade for the monoclonal antibody of methyl-2-oxazolidone (AMOZ) and detection residue of furaltadone through marker AMOZ.The present invention also provides application and enzyme-linked immunoassay method and the test kit application in residue of furaltadone through marker AMOZ detect of described monoclonal antibody in the enzyme linked immunological kit of preparation detection residue of furaltadone through marker AMOZ.
Above-mentioned purpose is achieved through the following technical solutions:
Can identify the monoclonal antibody of residue of furaltadone through marker 3-amino-5-beautiful jade for methyl-2-oxazolidone, it is secreted by hybridoma AMOZ.
Described hybridoma AMOZ, is deposited in Chinese Typical Representative culture collection center, and preserving number is CCTCCNO:C201188.
Described monoclonal antibody detects residue of furaltadone through marker 3-amino-5-beautiful jade for the application in the enzyme linked immunological kit of methyl-2-oxazolidone in preparation.
The test kit that comprises described monoclonal antibody.
Described test kit is the enzyme linked immunological kit for methyl-2-oxazolidone for detection of residue of furaltadone through marker 3-amino-5-beautiful jade.
The application of described test kit in residue of furaltadone through marker 3-amino-5-beautiful jade detects for methyl-2-oxazolidone.
Detect the enzyme-linked immunoassay method of residue of furaltadone through marker 3-amino-5-beautiful jade for methyl-2-oxazolidone, comprise the following steps:
(1) 3-terephthalaldehydic acid base-5 beautiful jade is obtained to immunogen (CPAMOZ-BSA conjugate) for methyl-2-oxazolidone (CPAMOZ) and bovine serum albumin (BSA) coupling;
(2) 3-terephthalaldehydic acid base-5 beautiful jade is obtained to coating antigen (CPAMOZ-OVA conjugate) for methyl-2-oxazolidone (CPAMOZ) and ovalbumin (OVA) coupling;
(3) utilize the immunogen of step (1) to prepare the hybridoma AMOZ that preserving number is CCTCC NO:C201188;
(4) prepare monoclonal antibody with the hybridoma AMOZ that preserving number is CCTCC NO:C201188;
(5) be coated with solid phase carrier with the coating antigen of step (2);
(6) sample pre-treatments: by testing sample acid treatment, add phenyl aldehyde ultrasonic derivative after, be extracted with ethyl acetate, get that ethyl acetate layer nitrogen dries up, normal hexane purifies and sample diluting liquid again dissolves and obtains determinand.
(7) determinand of step (6) is carried out to enzyme linked immunosorbent detection.
The application of described enzyme-linked immunoassay method in residue of furaltadone through marker 3-amino-5-beautiful jade detects for methyl-2-oxazolidone.
The invention has the beneficial effects as follows:
1. the monoclonal antibody that prepared by the present invention can be identified 3-phenyl aldehyde base-5 beautiful jade for methyl-2-oxazolidone (PAMOZ), and the derived products that the residual marker AMOZ that PAMOZ is Furaltadone reacts with phenyl aldehyde.
2, the present invention detects after not needing AMOZ to react with terephthalaldehydic acid again, but detects after reacting with phenyl aldehyde again, and detection efficiency is high.
3, monoclonal antibody, the enzyme linked immunological kit that prepared by the present invention and enzyme-linked immunoassay method detection sensitivity, the precision providing is high, accuracy good.
4. the haptens synthesis technique of the present invention's design is easy, and combined coefficient is high, and reagent used is the terephthalaldehydic acid that toxicity is less, less to the healthy harm of operator.The derivative reagent that sample pre-treatments is used is phenyl aldehyde, compares compared with other conventional derivative reagent Ortho Nitro Benzaldehydes, has advantages of that toxicity is little.
Brief description of the drawings
Fig. 1 is the canonical plotting that residue of furaltadone through marker AMOZ ELISA detects.Wherein, x axle is added with 100 the logarithmic value that adds concentration doubly and is drawn, and y axle is to measure the ratio (B/B of detection OD value in hole and zero medicine hole under each drug level
0) draw.
Embodiment
Below by embodiment, the invention will be further described, but do not limit the present invention.
Embodiment 1: antigen synthetic
1.1 haptens 3-terephthalaldehydic acid base-5 beautiful jades are synthetic for methyl-2-oxazolidone (CPAMOZ)
In brown vial, add terephthalaldehydic acid 37.5mg, slowly add methyl alcohol to dissolving completely, magnetic agitation.Add residue of furaltadone through marker AMOZ 50mg, room temperature lucifuge magnetic agitation reaction 48h.8000rpm, the centrifugal supernatant that goes of 10min, methanol wash 3 times, removes supernatant, and precipitation is dried to obtain white product CPAMOZ.
Synthesizing of 1.2 immunogens (CPAMOZ-BSA conjugate)
Get CPAMOZ 0.0167g and be dissolved in 1ml N, in dinethylformamide (DMF), add N, N ' dicyclohexylcarbodiimide (DCC) 0.0137g and N-hydroxy-succinamide (NHS) 0.0072g, 4 DEG C of magnetic agitation are spent the night.5000rpm, 10min, 4 DEG C of centrifuging and taking supernatants are for subsequent use is A liquid.Take 0.070g BSA and be dissolved in 5ml0.01M PH9.5 carbonate buffer solution (CBS), adding DMF (DMF) 0.5ml stirring and dissolving for subsequent use is B liquid.Under magnetic agitation, A liquid dropwise adds in B liquid, and 4 DEG C of reactions are spent the night.5000rpm, 10min, 4 DEG C of centrifuging and taking supernatants, normal saline dialysis 3 days, changes dialyzate every day 3 times at 4 DEG C.
Synthesizing of 1.3 coating antigens (CPAMOZ-OVA conjugate)
Get CPAMOZ 0.0167g and be dissolved in 1mlDMF, add DCC0.0137g and NHS 0.0072g, 4 DEG C of magnetic agitation are spent the night.5000rpm, 10min, 4 DEG C of centrifuging and taking supernatants are for subsequent use is A liquid.Take 0.070g OVA and be dissolved in 5ml 0.01M PH9.5CBS, adding DMF0.5ml stirring and dissolving for subsequent use is B liquid.Under magnetic agitation, A liquid dropwise adds in B liquid, and 4 DEG C of reactions are spent the night.5000rpm, 10min, 4 DEG C of centrifuging and taking supernatants, normal saline dialysis 3 days, changes dialyzate every day 3 times at 4 DEG C.
1.4 competition haptens 3-phenyl aldehyde base-5 beautiful jades are synthetic for methyl-2-oxazolidone (PAMOZ)
Taking AMOZ and phenyl aldehyde as raw material, synthetic PAMOZ.Add ethanol 1.5ml at the bottle being placed on magnetic stirring apparatus, in stirring, add phenyl aldehyde 21ul, AMOZ40mg, room temperature lucifuge reaction 24 hours, centrifugal, washing with alcohol 3 times, dry, obtain white product PAMOZ.
Embodiment 2: the preparation of monoclonal antibody
The preparation of hybridoma cell strain: the method with reference in Xue Qingshan " philosophy and technique of vitro culture " Science Press calendar year 2001 version: the CPAMOZ-BSA conjugate immunity Balb/C mouse of preparing with embodiment 1, immune programme for children is: fundamental immunity is by after immunogen and isopyknic Freund's complete adjuvant emulsification, in the subcutaneous multi-point injection of mouse back, once at interval of 2 weeks booster immunizations later, use Freund's incomplete adjuvant emulsification instead, finally in merging first three day abdominal injection, reinforced immunological, antigen amount doubles, and does not add adjuvant.When fusion, one of the Balb/C mouse of the last reinforced immunological of learning from else's experience, eye socket sacrificed by exsanguination (collect serum, be positive serum), soaks 5min sterilization in volume ratio in the alcohol that is 75%.
By 3 ~ 5 × 10
7sP2/0 myeloma cell and immune mouse spleen cell suspension join in 50mL centrifuge tube, mix the centrifugal 5min of 1500r/min.Abandon supernatant, and blot with the filter paper of sterilizing.Knock gently the pipe end, make pipe floor cells loosening.Centrifuge tube is placed in to 37 DEG C of water-baths, slowly adds 50% polyoxyethylene glycol (PEG) 0.8mL of pre-temperature to 37 DEG C in 1min, limit edged stirs with pipette tip gently, adds rear continuation and stirs 30sec, leaves standstill 1min.
Slowly add the RPMI-1640 basal liquid 10mL of 37 DEG C of pre-temperature.Concrete grammar is: 1min dropwise adds 1mL, and 2min adds 2mL, slowly adds afterwards remaining RPMI-1640 basal liquid, limit edged jog centrifuge tube.Slowly add 40mLRPMI-1640 basal liquid, after adding, slowly turn upside down and mix, the centrifugal 5min of 1500r/min.Abandon supernatant, 10mL dropper is drawn containing feeder cell HAT substratum, slowly drips along tube wall, by the slow mechanical stirring of dropper, slowly draws fused cell, approaches feeder cell liquid level and drips, and mechanical stirring mixes.Be inoculated in 6 96 well culture plates, approximately 150 μ L/ holes, are placed in 37 DEG C, 5%CO
2in cell culture incubator, cultivate.
Count 0d from merging the same day, after 3d, each culture hole drips 1 HAT substratum, sucks regularly the substratum of l/2 volume after 5d every 2d, changes to equivalent HT substratum.4d after fusion, starts fused cell to carry out tracing observation, mark and record the culture hole of Growth of Hybridoma Cell, and calculate fusion rate.6 ~ 7d after fusion, at the bottom of hole inner cell grows to hole 1/10～1/5 o'clock, gets culture supernatant, with indirect competitive ELISA method screening positive cell hole.Coating antigen concentration is 10mg/L.Each cell cultures hole arranges 0 hole and 200 μ g/L medicine holes, adds 50 μ L culture supernatant to carry out indirect competitive ELISA detection in every hole.
Select 4 ~ 6 supernatants to detect to be strong positive and only have the hole of 1 ~ 2 good colony growth of form, carry out subclone with limiting dilution assay.Through 3～4 time clonings, finishing screen is selected the monoclonal hybridoma strain of secreting specificity antibody.Applicant was this hybridoma called after AMOZ, and delivered Chinese Typical Representative culture collection center (CCTCC) preservation that is positioned at Wuhan City, Hubei Province Wuhan University on September 8th, 2011, and its preserving number is CCTCC NO:C201188.
The preparation of ascites monoclonal antibody and qualification: only within first 7 days, get Balb/c number of mice in inoculation, every mouse peritoneal injection 0.5ml Freund's incomplete adjuvant carries out pre-treatment.Suspending with RPMI – 1640 basic mediums is the cell of the hybridoma AMOZ enlarged culturing of CCTCC NO:C201188 by preserving number, and cell count is adjusted to 1 × 10
6individual/mL, every mouse peritoneal inoculation 0.5ml.Treat that mouse web portion obviously expands, spiritual variation, the dying ascites that gathers when motionless.According to literature method (Zhu Liping, Chen Xueqing. immunology common experimental method. Beijing: People's Medical Officer Press, 2000), purifying obtain monoclonal antibody.Adopt monoclonal antibody the present invention being obtained purchased from the mouse source monoclonal antibody hypotype identification kit (Mouse Mab Isotyping Test Kit) of ROCKLAND company to carry out hypotype qualification, result is mouse IgG 1 hypotype.
The foundation of embodiment 3:ELISA detection method
The optimization of 3.1ELISA condition
Accurately draw the CPAMOZ-OVA coating antigen working fluid 100 μ L that diluted in each hole, each concentration bag a line, in 4 DEG C of refrigerator overnight.Take out enzyme plate, every hole adds washings (PBST) 250 μ L in each hole, and cradle vibrate 1min, gets rid of clean washings, on thieving paper (or clean towel), pats dry.Repeated washing 2 times.Accurately draw confining liquid (1%OVA) 250 μ L in each hole, 37 DEG C of incubators are hatched 2h.Get rid of clean confining liquid, on thieving paper, pat dry.Accurately draw washings 250 μ L in each hole, cradle vibrate 1min, gets rid of clean washings, on thieving paper, pats dry.Repeated washing 2 times.
Get the enzyme plate being coated with, every hole adds the PBS damping fluid of 50 μ L 20mM pH7.4, the more capable primary antibodie 50 μ L that add same concentration to dilute of every row, 37 DEG C reaction 30min, wash 3 times, pat dry.Every hole adds the goat anti-rabbit igg working fluid 100 μ L of the HRP mark of 1:5000,37 DEG C reaction 30min, wash 3 times, pat dry.Add the each 100 μ L of substrate (A:B=1:100), 37 DEG C of lucifuge reaction 15min, by 50 μ L stop buffer termination reactions, measure light absorption value in 450nm.
Determining of the best coating antigen concentration of table 1
| 6400 | 12800 | 25600 | 51200 | 102400 | 204800 | |
| 16 | 3.796 | 3.775 | 3.703 | 3.204 | 2.339 | 1.575 |
| 8 | 3.914 | 3.802 | 3.617 | 3.193 | 2.288 | 1.494 |
| 4 | 3.42 | 3.704 | 3.648 | 3.046 | 2.251 | 1.456 |
| 2 | 3.808 | 3.642 | 3.423 | 2.73 | 2.049 | 1.313 |
| 1 | 3.668 | 3.578 | 3.35 | 2.677 | 1.801 | 1.109 |
| 0.5 | 3.595 | 3.419 | 3.006 | 2.205 | 1.514 | 0.901 |
| 0.25 | 3.366 | 2.991 | 2.538 | 1.801 | 1.115 | 0.602 |
| 0.125 | 2.82 | 2.324 | 1.868 | 1.305 | 0.728 | 0.439 |
As shown in table 1, determine that 0.25mg/L is the best effort concentration of coating antigen.With the coated concentration coated elisa plate of 0.25 μ g/mL, antibody dilution equal difference is designed to 1:40000,1:45000,1:50000,1:60000, carry out indirect competitive ELISA, drawing standard curve, calculates IC
50(in table 2).From table 2, determine that antibody optimum dilution degree is 1:50000.
Table 2 optimum antibody extent of dilution is optimized
| Antibody dilution (1: X) | " 0 " hole OD value | IC 50(μg/L) |
| 40000 | 2.108 | 1.02 |
| 45000 | 1.934 | 0.98 |
| 50000 | 1.825 | 0.89 |
| 60000 | 1.730 | 0.92 |
The foundation of 3.2 typical curves
To compete haptens 3-phenyl aldehyde base-5 beautiful jade and be mixed with for methyl-2-oxazolidone (PAMOZ) mother liquor of 1mg/mL with DMF, then with PBS, it is diluted to successively to 5 concentration such as 8.1,2.7,0.9,0.3,0.1,0 μ g/L, carry out indirect competitive ELISA, drawing standard curve, calculates IC
50.As shown in Figure 1, the regression equation of typical curve of the present invention is y=-0.615x+1.9538, index of correlation r=0.9961, IC
50value is 0.848 ± 0.0757 μ g/L (n=5), and linearity range is 0.1～8.1 μ g/L.
3.3 specificitys are selected itrofurans medicine prototype and metabolite and analog, substitute PAMOZ, measure its typical curve, and calculate IC
50, calculate cross reacting rate.Result is as shown in table 3, and this monoclonal antibody has specificity to PAMOZ.
Table 3 test kit cross reacting rate of the present invention
| Medicine name | IC 50(μg/L) | Cross reacting rate (%) |
| PAMOZ | 0.84 | 100 |
| Furaltadone | 17.9 | 4.68 |
| AMOZ | ＞1000 | ＜0.1 |
| AHD | ＞1000 | ＜0.1 |
| SEM | ＞1000 | ＜0.1 |
| CBA | ＞1000 | ＜0.1 |
| CPAOZ | ＞1000 | ＜0.1 |
| CPAHD | ＞1000 | ＜0.1 |
| CPSEM | ＞1000 | ＜0.1 |
| Nifurazolidone | ＞1000 | ＜0.1 |
| AOZ | ＞1000 | ＜0.1 |
| Nitrofural | ＞1000 | ＜0.1 |
| Furadantin | ＞1000 | ＜0.1 |
Embodiment 4: the assembling of ELISA detection kit of the present invention
4.1 test kit moietys
(1) be coated with the enzyme plate of coating antigen CPAMOZ-OVA conjugate;
(2) 6 bottles of PAMOZ standard solutions, concentration is respectively 0,0.1,0.3,0.9,2.7,8.1 μ g/L;
(3) hybridoma AMOZ monoclonal antibody working fluid;
(4) the sheep anti-mouse igg antibody working fluid of horseradish peroxidase (HRP) mark;
(5) concentrated phosphoric acid salt buffer: NaCl 80.0g, KH
2pO
42.0g, Na
2hPO
412H
2o 29.0g, KCl 2.0g, adds distilled water to 1000mL;
(6) concentrated cleaning solution: NaCl 80.0g, KH
2pO
42.0g, Na
2hPO
412H
2o 29.0g, KCl 2.0g, Tween-205mL, adds distilled water to 1000mL
(7) substrate mixed solution: accurately draw substrate B liquid (purchased from Fei Yuan Science and Technology Ltd. of Wuhan City) 10mL, add 100 μ L substrate A liquid (purchased from Fei Yuan Science and Technology Ltd. of Wuhan City), mix, now with the current.
(9) stop buffer: 2mol/L sulphuric acid soln.
The preparation of 4.2 enzyme plates
(1) coated: CPAMOZ – OVA to be diluted to 0.25 μ g/mL coating antigen solution with carbonate buffer solution, accurately to draw 100 μ L coating antigen solution in each enzyme mark hole, be placed horizontally at wet box, hatch 12h for 4 DEG C.
(2) wash plate: throw away enzyme plate endoperidium original solution, pat dry, accurately draw 250 μ L washingss in each enzyme mark hole, leave standstill after 30s, throw away washings, on thieving paper, pat dry; Repeated washing 3 times.
(3) sealing: accurately draw 250 μ L confining liquids in each enzyme mark hole, level is placed in wet box, and 37 DEG C of incubators are hatched 2h.
(4) wash plate: throw away confining liquid, accurately draw 250 μ L washingss in each enzyme mark hole, leave standstill after 30s, throw away washings, on thieving paper, pat dry; Repeated washing 3 times.
(5) dry: after patting dry on thieving paper; Enzyme plate is inverted in 37 DEG C of incubators and is dried 0.5h.
(6) encapsulation: enzyme plate packs aluminium foil bag into after drying together with siccative, encapsulates with vacuum packaging machine.Described siccative is silica gel or Calcium Chloride Powder Anhydrous one wherein.
Embodiment 5: test kit residual application of AMOZ in detection edible animal tissue
5.1 reagent preparations
Washings: by the concentrated cleaning solution providing in test kit with using after 10 times of dilutions of distilled water.
Sample diluting liquid: accurately take NaCl 8.0g, KH
2pO
40.2g, Na
2hPO
4.12H
2o 2.9g, KCl 0.2g, in 500mL beaker, adds a small amount of distilled water and dissolves, and distilled water is settled to 1000mL.
1M hydrochloric acid: accurately draw concentrated hydrochloric acid 8.2ml, distilled water is settled to 100mL.
10mM benzaldehyde solution preparation: accurately take phenyl aldehyde 1.06g, methanol constant volume is to 1000mL.
0.1M K
2hPO
4solution: accurately take K
2hPO
43H
2o 22.8g, adds a small amount of distilled water and dissolves, and distilled water is settled to 1000mL.
1M NaOH: accurately take NaOH 4g, add a small amount of distilled water and dissolve, distilled water is settled to 100mL.
The preparation of substrate mixed solution: according to each institute expense, get appropriate substrate A liquid and B liquid and mix in the ratio of 1:100, now with the current.
5.2 tissue sample processing
(1) tissue sample that takes 2.00 ± 0.02g homogeneous, in 50mL tool plug centrifuge tube, adds 4mL distilled water, 1M hydrochloric acid soln 0.5mL; With 10mM phenyl aldehyde 200uL, whirlpool concussion 1min, 50 DEG C of ultrasonic 2h;
(2) add respectively 0.1M K
2hPO
45mL, 1M NaOH 0.5mL; With the ethyl acetate of 6mL, vortex 2min.The at room temperature above centrifugal 10min of (20-25 DEG C) 4000r/min, the ethyl acetate of taking out 1.5mL dries up in 50 DEG C of nitrogen in another clean container;
(3) with the dry thing of 1mL n-hexane dissolution, then add 1mL PBS, vortex 2min, the above centrifugal 10min of (20-25 DEG C) 4000r/min under room temperature;
(4) take off layer water 50uL for analyzing.(extension rate: 2).
5.3ELISA measures program
(1) take out test kit, balance, to room temperature, is inserted micropore frame by the hole bar of enough standard substance and sample quantity used.
(2) add PAMOZ standard solution or sample liquid 50 μ L in micropore separately; Standard substance and sample do two parallel laboratory tests, record the position of standard substance and sample.Add antibody liquid 50 μ L to each hole, fully mix; Level is put in wet box, hatches 60min for 37 DEG C.
(3) get rid of liquid in clear opening, on thieving paper, pat dry.Accurately draw washings 250 μ L in each hole, leave standstill 30s left and right, get rid of clean washings, on thieving paper, pat dry.Repeated washing 4 times.
(4) the sheep anti-mouse igg antibody working fluid 100 μ L that add horseradish peroxidase (HRP) mark, to each hole, fully mix; Level is put in wet box, hatches 60min for 37 DEG C.
(5) get rid of liquid in clear opening, on thieving paper, pat dry.Accurately draw washings 250 μ L in each hole, leave standstill 30s left and right, get rid of clean washings, on thieving paper, pat dry.Repeated washing 5 times.
(6) add substrate mixed solution 100 μ L to each hole, fully mix; Level is put in wet box, hatches 15min for 37 DEG C.
(7) add stop buffer 50 μ L to each hole; Fully mix; Light absorption value is measured at the inherent 450nm of 30min place.
5.4 results are judged
The mean value of PAMOZ standard solution or sample liquid light absorption value is multiplied by 100% again divided by the light absorption value of " 0 " standard orifice, is inhibiting rate.Within the scope of 0.1 ~ 8.1 μ g/L, taking inhibiting rate as ordinate zou, the logarithm of concentration of standard solution is X-coordinate drawing standard curve, obtains regression equation.According to the inhibiting rate of formula 1 calculation sample, by inhibiting rate substitution regression equation, calculate mensuration concentration, be multiplied by dilution factor, be the residual concentration of AMOZ in sample.
Embodiment 6: the sensitivity of test kit of the present invention, precision, accuracy
The sensitivity of 6.1 test kits of the present invention
Sensitivity index using lowest detectable limit as test kit of the present invention.Get 20 parts of blank tissue samples, carry out ELISA detection, measure OD value, calculate the mean value of blank sample OD value
will
on substitution typical curve, find corresponding concentration (C), and calculate standard deviation (SD).Calculate Z value according to formula Z=C+3 × SD, the lowest detectable limit (LOD) that this is method for organizing, the results are shown in Table 4.
The lowest detectable limit of table 4AMOZ in various animal tissuess sample
| Tissue sample | Blank sample is measured mean value (μ g/kg, n=20) | SD(n=20) | LOD(μg/kg) |
| Pork | 0.21 | 0.02 | 0.27 |
| Shrimp | 0.23 | 0.01 | 0.26 |
| The flesh of fish | 0.25 | 0.01 | 0.28 |
| Honey | 0.28 | 0.01 | 0.31 |
The precision of 6.2 test kits of the present invention
PAMOZ standard substance are diluted to 0,0.1,0.3,0.9,2.7,8.1 μ g/L, 3 parallel holes of each concentration, according to indirect competitive ELISA method replication 5 times, its typical curve equation of OD value substitution corresponding each standard substance concentration is obtained to the measured value that ELISA detects, with the variation coefficient between the plate inner panel of standard substance concentration determination value calculating indirect competitive ELISA typical curve, the precision that judges test kit with this, the results are shown in Table 5.Visible, the precision of test kit of the present invention is good.
The variation coefficient in the plate of table 5 test kit of the present invention and between plate
The accuracy of 6.3 test kits of the present invention
In homogeneous good various tissue samples, add AMOZ standardized solution, make its final concentration be respectively 0.5 μ g/kg and 1 μ g/kg.5 Duplicate Samples of each concentration, then carry out sample preparation, and ELISA measures drug level, repeat 3 batches, calculate recovery rate, and the rate of recovery is calculated by formula 2, and calculates batch interior and interassay coefficient of variation, the results are shown in Table 6～9.Visible, the interpolation of test kit of the present invention is reclaimed all 60% ~ 130%, and batch variation < 20% in crowd, shows that accuracy is good.
In table 6 pig muscle, AMOZ adds the rate of recovery and the variation coefficient
In table 7 flesh of fish, AMOZ adds the rate of recovery and the variation coefficient
In table 8 shrimp, AMOZ adds the rate of recovery and the variation coefficient
In table 9 honey, AMOZ adds the rate of recovery and the variation coefficient
Claims (2)
1. an enzyme-linked immunoassay method for detection residue of furaltadone through marker 3-amino-5-morpholino methyl-2-oxazolidone of non-diagnostic purpose, is characterized in that: comprise the following steps:
(1) 3-terephthalaldehydic acid base-5-morpholino methyl-2-oxazolidone and ovalbumin coupling are obtained to coating antigen;
(2), with being deposited in Chinese Typical Representative culture collection center, preserving number is that the hybridoma AMOZ of CCTCC NO:C201188 prepares monoclonal antibody;
(3) be coated with solid phase carrier with the coating antigen of step (1);
(4) sample pre-treatments: by testing sample acid treatment, add phenyl aldehyde ultrasonic derivative after, be extracted with ethyl acetate, get that ethyl acetate layer nitrogen dries up, normal hexane purifies and sample diluting liquid again dissolves and obtains determinand;
(5) determinand of step (4) is carried out to enzyme linked immunosorbent detection.
2. the application of enzyme-linked immunoassay method claimed in claim 1 in the detection of the non-diagnostic purpose of residue of furaltadone through marker 3-amino-5-morpholino methyl-2-oxazolidone.
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| CN103954749B (en) * | 2014-04-17 | 2015-11-11 | 华南农业大学 | A kind of tachysynthesis detection method for direct-detection AMOZ AMOZ |
| CN103951630B (en) * | 2014-04-17 | 2016-06-01 | 华南农业大学 | The tree-shaped haptens of a kind of direct-detection Furaltadone metabolite AMOZ, tree-shaped antigen and application thereof |
| CN105907723A (en) * | 2016-05-31 | 2016-08-31 | 江南大学 | Monoclonal antibody hybridoma cell strain YH4 resisting furaltadone metabolite AMOZ and application thereof |
| CN106645687A (en) * | 2016-11-09 | 2017-05-10 | 百奥森（江苏）食品安全科技有限公司 | Kit for detecting furaltadone metabolites in food |
| CN108047156B (en) * | 2017-12-26 | 2020-08-07 | 华南农业大学 | 5-methylmorpholine-3-amino-2-oxazolidinyl ketone antigen with spiro structure, antibody, preparation and application |
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| Title |
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| "呋喃它酮代谢物AMOZ单克隆抗体的制作与免疫学特性鉴定";范国英等;《中国畜牧兽医学会2010年学术年会——第二届中国兽医临床大会论文集(下册)》;20100815;第1616-1619页 * |
| 范国英等."呋喃它酮代谢物AMOZ单克隆抗体的制作与免疫学特性鉴定".《中国畜牧兽医学会2010年学术年会——第二届中国兽医临床大会论文集(下册)》.2010,第1616-1619页. |
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